Difference between revisions of "Team:UNIK Copenhagen/Results"
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| − | <p style="font-size:10.5px; margin: 4px 0px | + | <p style="font-size:10.5px; margin: 4px 170px 0px 120px"> Figure 4: Fluorescence microscopy pictures of P. patens transformed with our genetic constructs. A) A moss protoplast transformed with the antifreeze construct. B) A moss protoplast transformed with our STS construct. C) A moss protoplast transformed with a vector expressing YFP. A positive control. D) WT moss. A negative control. 1) Bright field picture. 2) Filter showing autoflouorescence (red) and YFP-expression (green). 3) Filter showing only YFP-expression (green).</p style> |
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| − | <p> | + | <p>Our transformed moss protoplasts were then moved to to PhyB-plates containing kanamycin (50 mg/ml) and were left to grow for a few weeks. After a few weeks we observed growing clumps of moss on for both constructs. Many of the growing moss clumps did not express visible YFP under fluorescent microscopy and we hypothesize that this may be because only Piece A and perhaps Piece B/C has integrated into the moss genome or perhaps the levels of YFP expression is just to low to be visible. We did see a clump of antifreeze moss with clear YFP expression (fig. 4). |
| + | This suggest varying degree of stable integration of our gene constructs. Our moss was able to grow from protoplasts to full clumps media containing kanamycin, which suggests that the nptII-resistance cassette provides Physcomitrella Patens with resistance to kanamycin. To further validate this, we outlined an additional experiment. | ||
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| − | + | <p style="font-size:10.5px; margin: 4px 170px 0px 120px"> Figure 5: A clump of transformed P. Patens a few weeks after transformation showing YFP-expression, grown on kanamycin containing plates (50 mg/ml). A) Bright field picture. B) Filter showing autoflouorescence (red) and YFP-expression (green). <br> | |
| − | < | + | C) Filter showing only YFP-expression (green). |
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| + | <h2>Considerations for replicating the experiments</h2> | ||
| + | Here you can read about our successes and failures over your summer. | ||
| + | <h2>Future plans</h2> | ||
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Revision as of 13:35, 6 September 2015
Project Results
A few days after transformation we observed the moss protoplasts under a fluorescent microscope and saw YFP expression in moss protoplats for the antifreeze and the STS construct (fig. 4A and 4B). This confirms the that our transformation was a success and highly suggests that our genes of interest, antifreeze and STS, are expressed. It demonstrated that our construct design works and that moss can combine different DNA pieces with matching overhangs using homologous recombination.
Figure 4: Fluorescence microscopy pictures of P. patens transformed with our genetic constructs. A) A moss protoplast transformed with the antifreeze construct. B) A moss protoplast transformed with our STS construct. C) A moss protoplast transformed with a vector expressing YFP. A positive control. D) WT moss. A negative control. 1) Bright field picture. 2) Filter showing autoflouorescence (red) and YFP-expression (green). 3) Filter showing only YFP-expression (green).
Our transformed moss protoplasts were then moved to to PhyB-plates containing kanamycin (50 mg/ml) and were left to grow for a few weeks. After a few weeks we observed growing clumps of moss on for both constructs. Many of the growing moss clumps did not express visible YFP under fluorescent microscopy and we hypothesize that this may be because only Piece A and perhaps Piece B/C has integrated into the moss genome or perhaps the levels of YFP expression is just to low to be visible. We did see a clump of antifreeze moss with clear YFP expression (fig. 4). This suggest varying degree of stable integration of our gene constructs. Our moss was able to grow from protoplasts to full clumps media containing kanamycin, which suggests that the nptII-resistance cassette provides Physcomitrella Patens with resistance to kanamycin. To further validate this, we outlined an additional experiment.
Figure 5: A clump of transformed P. Patens a few weeks after transformation showing YFP-expression, grown on kanamycin containing plates (50 mg/ml). A) Bright field picture. B) Filter showing autoflouorescence (red) and YFP-expression (green).
C) Filter showing only YFP-expression (green).